CN109180571B

CN109180571B - Bipyridine derivative, and synthesis method and application thereof

Info

Publication number: CN109180571B
Application number: CN201811257856.1A
Authority: CN
Inventors: 吴俊清; 郭廷翘; 章健; 吴冠英
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-10-26
Filing date: 2018-10-26
Publication date: 2021-08-13
Anticipated expiration: 2038-10-26
Also published as: CN109180571A

Abstract

The invention provides a bipyridine compound, and a cryptate compound product of the bipyridine compound is a fluorescent material which shows potential application prospects in the fields of luminescence, illumination, fluorescent probes and the like, can completely change the defects of the conventional dissociation-enhanced time-resolved fluorescence immunoassay (DELFLA), and realizes high-sensitivity, large-flux and full-automatic time-resolved immunofluorescence detection.

Description

Bipyridine derivative, and synthesis method and application thereof

Technical Field

The invention belongs to the technical field of fluorescent probes, and particularly relates to a bipyridine derivative and application thereof.

Background

With the rapid development of medicine and life science, the requirement for detecting trace substances is higher and higher, so that new detection technologies are continuously developed. A novel analysis method was first proposed by Soini and Kojola in the last 70 th century by Time-Resolved fluoroimmunoassay (TRFIA). Due to the fact that the lanthanide ion chelate has large Stokes shift, nonspecific fluorescence interference is eliminated through the selective filter plate; and because the lanthanide chelating agent can emit long-life fluorescence, the influence of short-life fluorescence background can be eliminated by a time-resolved fluorescence analysis technology, the signal-to-noise ratio is greatly improved, and the detection sensitivity is greatly improved. Because the sensitivity of immunoassay can be greatly improved, the TRFIA technology replaces a high-sensitivity radioimmunoassay method, and has shown a huge application prospect in the technical field of international ultramicro analysis. However, the current TRFIA technology mainly adopts Dissociation Enhanced Lanthanide Fluorescence Immunoassay (DELFIA), lanthanide ions marked by the system are not fluorescent, and a new lanthanide chelate is formed after an enhancing solution is added to emit strong fluorescence. During the formation of the new lanthanide chelate system, lanthanide ions have been dissociated from the label, homogeneously dissociated in the enhancing fluid, and lost their labeling effect. Therefore, the system can not be applied to a full-automatic random detection system of a time-resolved immunofluorescence full-automatic detection system (short for time-resolved random system) which needs to use a specific labeled tracer. For this reason, it is necessary to develop a novel bifunctional chelating agent.

Lanthanide series complex cryptate compounds are fluorescent materials which show potential application prospects in the fields of luminescence, illumination, fluorescent probes and the like. Cryptates are formed by incorporating a cation into a steric cage. The cage can collect light energy and then transfer the energy to the lanthanide of the core. The nature of the macrocycle facilitates close association with the lanthanide, and such an unbreakable connection can form an exceptionally robust complex. The time-resolved stochastic system developed based on lanthanide cryptate can completely change the defects of the conventional dissociation-enhanced time-resolved fluoroimmunoassay (DELFLA) method, and realize high-sensitivity, high-flux and full-automatic time-resolved immunofluorescence detection.

The bipyridine substance can react with lanthanide series Eu due to the chelating action and the existence of electron donating capability³⁺Form stable complexes, which can thus act as central building blocks for organic ligands, which react further to form Eu³⁺Crypts, serving to sensitize Eu³⁺The effect of luminescence and connection of the biomolecule to be detected is that the compound has better performance. In recent years, many compounds and intermediates of this kind have been synthesized, and exhibit excellent fluorescence properties such as high lanthanide quantum yield and kinetic stability.

The lanthanide compound selected by the patent "a lanthanide compound and a preparation method and application thereof" (CN 105218570B) is an unstable amide bond, and in the course of its synthesis from A8 to a9, products with single-branch, double-branch, three-branch, four-branch, five-branch and six-branch side chains are generated, and when one branch is not generated, products with the same branch number and different positions combined in 6 spatial positions may be generated, so that such a great number of byproducts are obtained, the yield of single-branch chains is inevitably low, and the chemical structures of various byproducts are not very different, which makes the product separation and purification very difficult, and directly affects the purity of the target product and the application in the immunoassay method.

The cryptate described in US7087384B2 is mainly used in imaging, and therefore it does not consider the quantitative relationship of the linker protein to which a compound is attached, and two linker protein positions are designed for synthetic convenience. The patent can not ensure that each fluorophore is linked with an antigen and an antibody, reduces the tracing accuracy of 1 to 1 as a fluorescent marker, and ensures that the fluorescent marker can not distinguish and identify whether one antigen or antibody participates in immune reaction or 2 antigens simultaneously participate in immune reaction, so the application of the fluorescent marker in immunodiagnosis is limited. In addition, the patent has the defects that the branch arms on the binding protein are short, the self hydrophilicity exists or the point position influences the binding of the coupling, and even influences the immunoreaction specificity of the coupling protein.

Disclosure of Invention

Aiming at the problem that the shortage of a novel bifunctional neoplasms intermediate greatly limits the popularization of a TRFIA technology, the invention provides a bipyridyl compound, and a cryptate compound product of the bipyridyl compound is a fluorescent material which shows potential application prospects in the fields of luminescence, illumination, fluorescent probes and the like.

In order to realize the purpose of the invention, the invention adopts the technical scheme that:

a bipyridine derivative having a chemical formula:

r1 and R2 are both Br;

or,

r1, R2 are each attached to two linkages of the following cryptates, forming lanthanide cryptates:

the bipyridine derivative has the following structure:

the synthetic route of the bipyridyl compound is as follows:

the method comprises the following steps:

A. epoxidizing a pyridine of the compound (4) with a peroxide to produce a compound (5); B. nitrifying the compound (5) with sulfuric acid and nitric acid to produce a compound (6);

C. converting the nitro group on compound (6) to bromine with acetyl bromide to produce compound (7); D. reducing compound (7) to compound (8) with phosphorus tribromide;

E. condensing the compound (8) with 3-aminopropanol to produce a compound (9);

F. the compound (9) is subjected to acylation reaction to generate a compound (10);

G. oxidizing the compound (10) with m-CPBA to compound (11);

H. reacting the compound (11) with acetic anhydride to produce a compound (12);

I. hydrolyzing the compound (12) to a compound (13);

J. compound (13) is brominated to produce compound (1).

Preferably, the peroxide of step A is m-CPBA.

Preferably, the compound for acylation reaction of the compound (9) in the step F is benzoyl chloride, benzyl chloroformate or tert-butyl chloroformate.

Preferably, the brominating reagent for said J step is phosphorus tribromide or hydrogen bromide.

The bipyridine derivative has the following structure:

the synthesis method of the cryptate compound comprises the following steps:

A. dissolving the compound (2) in a solvent containing lithium carbonate, reacting with the compound (1), stirring, cooling in an ice bath, filtering, evaporating the filtrate to dryness, and performing reverse phase chromatography on the residue to obtain a cryptic product (3);

the structure of compound (2) is:

the structure of compound (3) is:

B. adding europium trichloride and the compound (3) into a solvent, refluxing, evaporating the solvent, dissolving in an acid solution, stirring at room temperature, then evaporating excess acid under reduced pressure, concentrating to dryness, and performing reverse phase chromatography to obtain the compound (3').

The solvent is ethanol, methanol, diethyl ether, acetonitrile, dimethyl sulfoxide or dioxane, and the preferable solvent is acetonitrile.

The acid solution is trifluoroacetic acid.

The invention relates to a time-resolved fluorescence detection kit for a cryptate (3').

The cryptate compound (3') can be connected with protein to form a tracer detection object, and the cryptate compound and the protein marked by the magnetic microspheres are assembled into a detection kit together, so that the detection kit is applied to a time-resolved immunoassay full-automatic detection system.

The invention has the beneficial effects that:

1. the cryptate is connected with protein groups of organisms through amino groups on the exocyclic side chain, and the cryptate of the invention only has the linkage of a single side chain, thereby improving the sensitivity and specificity of immunoassay.

2. The invention directly connects the connexin group in advance, constructs the single-branch structure firstly, protects the connexin group, ensures that the capacity of the final target product of the coupled protein is not influenced in the synthesis process, and avoids the defects of the patent CN 105218570B.

3. The bipyridyl compound (1) is synthesized for the first time, the arm length of a cavernous compound structure obtained by taking the bipyridyl compound (1) as a raw material is increased by more than 1 time, the spatial interference of the self potential or hydrophilicity of a marker on a combined coupled antigen or antibody can be effectively reduced, the immune characteristic of the coupled protein is exerted, and the detection tracing precision in immunodiagnosis is greatly improved.

4. The synthetic method of the bipyridyl compound (1) adopts a three-step bromination method of oxidizing bipyridyl into N-oxide and then brominating, so that the bromination product is single, the side reaction products are few, the purification is easy, and the yield is high.

Drawings

FIG. 1 is an NMR chart of the compound (1) of the present invention.

FIG. 2 is a working curve of cryptate (3') further synthesized with compound (1) of the present invention for use in the determination of thyrotropin.

Detailed Description

In order to more clearly and specifically illustrate the technical solution of the present invention, the present invention is further described by the following embodiments. The following examples are intended to illustrate the practice of the present invention and are not intended to limit the scope of the invention.

Example 1

The synthetic route of compound (1) is as follows:

the synthesis method comprises the following steps:

A. epoxidizing a pyridine of the compound (4) with m-CPBA to produce a compound (5);

B. nitrifying the compound (5) with sulfuric acid and nitric acid to produce a compound (6);

C. converting the nitro group on compound (6) to bromine with acetyl bromide to produce compound (7);

D. reducing compound (7) to compound (8) with phosphorus tribromide;

E. condensing the compound (8) with 3-aminopropanol to produce a compound (9);

F. reacting the compound (9) with benzoyl chloride to generate a compound (10);

G. oxidizing the compound (10) with m-CPBA to compound (11);

H. reacting the compound (11) with acetic anhydride to reduce the N-oxide to produce a compound (12);

I. hydrolyzing the compound (12) to a compound (13);

J. compound (13) is brominated using phosphorus tribromide to give compound (1).

Example 2

This example is based on example 1:

said step a epoxidizes one pyridine of compound (4) with peroxybenzoic acid.

And the compound which is subjected to acylation reaction with the compound (9) in the step F is benzyl chloroformate.

Example 3

This example is based on example 1:

in the step A, one pyridine of the compound (4) is epoxidized with ammonium persulfate or the like.

The compound acylated with compound (9) in step F is tert-butyl chloride.

The brominating reagent used in step J is hydrogen bromide.

Example 4

Synthesis of 6,6 '-dimethyl-2, 2' -bipyridine-N-oxide (5)

6,6 '-dimethyl-2, 2' -bipyridine (4) (39.4g,0.21mol) was dissolved in 1000ml of chloroform and m-chloroperoxybenzoic acid (49.0g,0.21mol, 75% wt) was added in small portions at 0 ℃. After the addition was completed, the mixture was stirred at room temperature for 4 hours. The reaction was monitored by TLC. The mixture was washed successively with a saturated sodium bicarbonate solution, brine, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to give a crude product. The crude product was purified by silica gel column chromatography (DCM: MeOH ═ 30:1) to give 6,6 '-dimethyl-2, 2' -bipyridine-N-oxide (30.0g, 70%) as a colorless oil. LCMS (ESI) M/z 201(M + H) +; RT ═ 1.6 min.

Example 5

Synthesis of 4-nitro-6, 6 '-dimethyl-2, 2' -bipyridine-N-oxide (6)

To a mixture of sulfuric acid (135mL) and nitric acid (100mL) was added 6,6 '-dimethyl-2, 2' -bipyridine-N-oxide (5) (25.0g,0.12mol), and the mixture was stirred at 100 ℃ for 1 hour. After the completion of the reaction was monitored by TLC, the mixture was poured into ice water, the pH was adjusted to 8 with 1N sodium hydroxide solution and sodium bicarbonate solution, and the precipitated precipitate was filtered and dried to give 4-nitro-6, 6 '-dimethyl-2, 2' -bipyridine-N-oxide (20.0g, 65%) as a yellow solid. LCMS (ESI) M/z 246(M + H) +; RT ═ 1.9 min.

Example 6

Synthesis of 4-bromo-6, 6 '-dimethyl-2, 2' -bipyridine-N-oxide (7)

To a solution of 4-nitro-6, 6 '-dimethyl-2, 2' -bipyridine-N-oxide (6) (15.0g,0.06mol) in acetic acid (120mL) was added acetyl bromide (65 mL). The mixture was stirred at 80 ℃ for 1.5 hours. After the completion of the reaction was monitored by TLC, the mixture was poured into ice water and the pH was adjusted to 8 with 1N sodium hydroxide solution and potassium hydroxide solid solution. The mixture was then extracted 3 times with ethyl acetate, and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. The crude product was chromatographed on NH silica gel (PE: EA ═ 10:1 to 2:1) to give 4-bromo-6, 6 '-dimethyl-2, 2' -bipyridine-N-oxide (5.0g, 29%) as a yellow solid. LCMS (ESI) M/z 279(M + H) +; RT ═ 1.7 min.

Example 7

Synthesis of 4-bromo-6, 6 '-dimethyl-2, 2' -bipyridine (8)

To a solution of 4-bromo-6, 6 '-dimethyl-2, 2' -bipyridine-N-oxide (7) in chloroform (100mL) was added phosphorus tribromide (11 mL). The mixture was stirred at room temperature for 15 minutes. After the completion of the reaction was monitored by TLC, the pH was adjusted to 8 with 1N sodium hydroxide solution and solid potassium hydroxide solution. The mixture was then extracted 3 times with dichloromethane, and the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give 4-bromo-6, 6 '-dimethyl-2, 2' -bipyridine (4.8g, 90%) as a brown solid. LCMS (ESI) M/z 263(M + H) +; RT ═ 1.0min.

Example 8

Synthesis of 4- (3-aminopropoxy) -6,6 '-dimethyl-2, 2' -bipyridine (9)

Potassium hydroxide (2.0g,36.64mmol) and DMSO (15mL) were combined and stirred at 60 ℃ for 1h, then 4-bromo-6, 6 '-dimethyl-2, 2' -bipyridine (8) (4.8g,18.32mmol) and 3-aminopropanol (2.8g,18.32mmol) were added and stirred at 60 ℃ overnight. After the completion of the reaction was monitored by TLC, water was added, extracted 3 times with ethyl acetate, washed successively with water, brine, dried over anhydrous sodium sulfate, and filtered to obtain a crude product. The crude product was chromatographed on NH silica gel (DCM: MeOH ═ 20:1 to 5:1) to give 4- (3-aminopropoxy) -6,6 '-dimethyl-2, 2' -bipyridine (2.9g, 61%) as a light yellow oil. LCMS (ESI) M/z 258(M + H) +; RT ═ 1.6 min.

Example 9

Synthesis of 4- (3-benzoylaminopropoxy) -6,6 '-dimethyl-2, 2' -bipyridine (10)

4- (3-Aminopropoxy) -6,6 '-dimethyl-2, 2' -bipyridine (9) (3.0g,11.67mmol) was dissolved in pyridine (20mL), and benzoyl chloride (1.8g,12.84mmol) was added. The mixture was stirred at room temperature for 20 minutes. After completion of the reaction was monitored by TLC, chloroform and water were added, and the aqueous layer was separated and extracted again with aluminum. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. The crude product was chromatographed on NH silica gel (DCM: MeOH ═ 20:1 to 10:1) to give 4- (3-benzoylaminopropoxy) -6,6 '-dimethyl-2, 2' -bipyridine (3.7g, 87%) as a white solid. LCMS (ESI) M/z 362(M + H) +; RT ═ 1.0min.

Example 10

Synthesis of 4- (3-benzoylaminopropoxy) -6,6 ' -dimethyl-2, 2 ' -bipyridine-N, N ' -dioxide (11)

4- (3-Benzoylaminopropoxy) -6,6 '-dimethyl-2, 2' -bipyridine (10) (1.0g,2.77mmol) was dissolved in 70mL of chloroform and m-chloroperoxybenzoic acid (3.2g,13.85mol, 75% wt) was added in portions at 10 ℃. After stirring at room temperature for 2 hours and monitoring the completion of the reaction by TLC, the reaction was successively washed with saturated sodium bicarbonate solution, brine, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to give a crude product. The crude product was purified by NH silica gel column chromatography (DCM: MeOH ═ 30:1 to 10:1) to give 4- (3-benzoylaminopropoxy) -6,6 ' -dimethyl-2, 2 ' -bipyridine-N, N ' -dioxide (750mg, 69%) as a white solid. LCMS (ESI) M/z394(M + H) +; RT ═ 1.6 min.

Example 11

Synthesis of 4- (3-benzoylaminopropoxy) -6,6 '-diacetoxymethyl-2, 2' -bipyridine (12)

4- (3-Benzoylaminopropoxy) -6,6 ' -dimethyl-2, 2 ' -bipyridine-N, N ' -dioxide (11) (1.5g,3.81mmol) and acetic anhydride (30mL) were mixed and refluxed for 15 minutes. After the completion of the reaction was monitored by TLC, the mixture was evaporated to dryness to give the crude product. The crude product was chromatographed on NH silica gel (DCM: MeOH ═ 30:1) to give 4- (3-benzoylaminopropoxy) -6,6 '-diacetoxymethyl-2, 2' -bipyridine (1.9g, 95%) as a yellow solid. LCMS (ESI) M/z 478(M + H) +; RT ═ 1.0min.

Example 12

Synthesis of 4- (3-benzoylaminopropoxy) -6,6 '-dimethylol methyl-2, 2' -bipyridine (13)

4- (3-Benzoylaminopropoxy) -6,6 '-diacetoxymethyl-2, 2' -bipyridine (12) (1.9g,3.98mmol) was dissolved in methanol (20mL), and 1N aqueous sodium hydroxide solution (2mL) was added. The mixture was stirred at room temperature for 1 hour. After the completion of the reaction was monitored by TLC, water was added and extracted 3 times with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to give the crude product. The crude product was chromatographed on NH silica gel (DCM: MeOH ═ 30:1 to 10:1) to give 4- (3-benzoylaminopropoxy) -6,6 '-dimethylolmethyl-2, 2' -bipyridine (800mg, 51%) as a yellow oil. LCMS (ESI) M/z394(M + H) +; RT ═ 2.3 min.

Example 13

Synthesis of 4- (3-benzoylaminopropoxy) -6,6 '-dibromomethyl-2, 2' -bipyridine (1)

4- (3-Benzoylaminopropoxy) -6,6 '-dimethylolmethyl-2, 2' -bipyridine (13) (200mg,0.51mmol) was dissolved in DMF (10mL) and phosphorus tribromide (0.1mL) was added at 0 ℃. The mixture was stirred at room temperature for 30 minutes. After completion of the reaction was monitored by TLC, it was poured into ice water. The pH was adjusted to 9 with 1N aqueous sodium hydroxide solution, and extracted 3 times with ethyl acetate. The organic layers were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure to give the crude product. The crude product was purified by NH silica gel column chromatography (DCM: MeOH ═ 150:1) to give compound (1) (80mg, 30%) as a white solid. LCMS (ESI) M/z 520(M + H) +; RT 3.2min hplc 96%, RT 4.9min 1H NMR (400MHz, CDCl3) δ 8.37(d, J7.1 Hz,1H),7.95(d, J2.3 Hz,1H),7.80(m,3H),7.48(m,4H),7.00(d, J2.3 Hz,1H),6.50(s,1H),4.62(s,2H),4.56(s,2H),4.31(t, J5.8 Hz,2H),3.72(dd, J12.4, 6.3Hz,2H), 2.26-2.16 (m,2H), as shown in fig. 1.

Example 14

The synthetic route for cryptate (3') of the present invention is as follows:

the synthesis method comprises the following steps:

A. dissolving the compound (2) in a solvent containing lithium carbonate, reacting with the compound (1), stirring, cooling in an ice bath, filtering, evaporating the filtrate to dryness, and performing reverse phase chromatography on the residue to obtain a compound (3);

Example 15

Synthesis of Compound (3)

25.3mg (3.18X 10-5mol) of Compound (2) (synthesized as described in US 7087384) and 27mg (3.65X 10-4mol) of lithium carbonate were mixed in 25ml of anhydrous acetonitrile and refluxed for 15 minutes under a nitrogen atmosphere. 16.06mg (3.1X 10-5mol) of compound (1) was added dropwise over 10 minutes, resulting in a suspension in a solution of anhydrous acetonitrile. Stirring was continued under these conditions for 23 hours, then cooled in an ice bath and filtered. The filtrate was evaporated to dryness and the residue was chromatographed on reverse phase using acetonitrile/water containing 1% TFA to give compound (3).

The anhydrous acetonitrile solvent can be replaced by ethanol, methanol, diethyl ether, dimethyl sulfoxide or dioxane.

Example 16

Synthesis of Compound (3')

12.8mg (3.5X 10-5mol) of europium trichloride hexahydrate and 17.46mg (1.51X 10-5mol) of compound (3) are added together to a solution in 10ml of anhydrous acetonitrile. The reaction mixture was refluxed for 3 hours under nitrogen blanket. Then cooled and the solvent evaporated. The residue was used directly in the next reaction.

The residue obtained in the above step was dissolved in 14ml of trifluoroacetic acid. The resulting homogeneous solution was stirred at room temperature for 4 hours, then the excess acid was evaporated under reduced pressure and concentrated to dryness. By reverse phase chromatography, eluting with 1% trifluoroacetic acid/acetonitrile in water, the europium chelate of the tetracarboxy propionyl amino compound (compound 3') was obtained.

Wherein the trifluoroacetic acid can be replaced by inorganic acid such as hydrochloric acid.

Example 17

Maleic acid imino group activation of cryptate (3')

To a solution of 16.04mg (1.28X 10-7mol) of the bicyclic cryptate (3') in 200. mu.l of 0.1M phosphate buffer solution having a pH of 7.0 was added dropwise a solution of 195. mu.g (4.45X 10-7mol) of the sodium salt of sulfonic succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC) in 168. mu.l of phosphate buffer solution with cooling in an ice bath. After the addition, the temperature of the medium was raised to 20-25 ℃ and the reaction was continued for 3 hours. Direct reverse phase chromatography, eluting with acetonitrile containing 1% trifluoroacetic acid, then afforded about 100 μ g of the title compound activated cryptate (14).

Example 18

Activated cryptate (14) labelled anti-human-TSH alpha-subunit monoclonal antibodies

1mg of anti-human-TSH alpha-subunit monoclonal antibody (purchased from Mitsubishi chem. Co.) was first dialyzed four times against a 0.05M boric acid buffer solution pH8.3 at 2-8 ℃. Taking out and adding 25mmol/L PTT (dithiothreitol), stirring at normal temperature for reaction for 25 minutes, and then adding 0.2-0.5 mg of activated cryptate (14) for reaction for 20 hours at low temperature.

Separating monoclonal antibody marked with cryptate and monoclonal antibody not marked by Sephadex G-50 column chromatography, distinguishing proteins with different molecular weights by using a protein instrument, and simultaneously detecting the proportion of cryptate marked to monoclonal antibody.

Example 19

The magnetic beads were coated with a monoclonal antibody directed against the beta subunit of human TSH.

1mg of anti-human TSH beta-subunit monoclonal antibody (purchased from Mitsubishi chem. Co.) was dialyzed with 0.05M boric acid buffer solution of pH8.0 at 2-8 ℃ for four times, and finally the volume was taken out to be not more than 1 ml.

Sucking 10ml of magnetic particles with 2 microns (solid content is 2%) of particle size and carboxyl on the surface at a certain position by using a magnet, sucking out supernatant, dissolving 5ml of 25mM MES buffer solution (pH7.4), adding 5mg of NHS (N-hydroxysuccinimide) activator and 2-5 mg of EDC (carbodiimide) activator after uniformly mixing, and fully stirring for 30 minutes; the activated magnetic beads were attracted to a magnet, the supernatant was aspirated, and 2ml of a boric acid buffer (pH8.2) was added thereto.

And (3) placing the dialyzed anti-human TSH beta-subunit monoclonal antibody into activated magnetic beads, carrying out low-temperature oscillation reaction for 24 hours, then sucking the activated magnetic beads by using a magnet, sucking out the supernatant, then injecting 10 microliters of 1% BSA (bovine serum albumin) blocking solution, carrying out oscillation blocking at room temperature for 12 hours, then sucking the supernatant by using the magnet, finally adding the diluent, and storing at the low temperature of 2-8 ℃.

Example 20

Preparation of thyrotropin kit

Preparing a TSH standard: TSH standard solutions are prepared, and the specific concentrations are 0, 0.09, 0.84, 4.4, 22.5 and 115mIU/L respectively.

The magnetic beads marked by the anti-human TSH beta-subunit monoclonal antibody prepared in the example 19 and the anti-human TSH alpha-subunit monoclonal antibody marked by the cryptate compound prepared in the example 18 are adjusted in concentration, the most appropriate proportion is determined, and then the magnetic beads marked by the anti-human TSH beta-subunit monoclonal antibody are combined with TSH standard products, wherein the reaction process comprises the steps of putting a certain amount of the magnetic beads marked by the anti-human TSH beta-subunit monoclonal antibody prepared in the example 15 into a tubular reactor, adding the standard products into the tubular reactor for reaction, separating the magnetic beads by using an electromagnetic field after reacting for 1 hour in a rotating magnetic field, taking away supernatant, cleaning, adding the anti-human TSH alpha-subunit monoclonal antibody marked by the cryptate compound prepared in the example 14, separating the magnetic beads by using the electromagnetic field after reacting for 1 hour in the rotating magnetic field, taking away supernatant, and cleaning. And (3) sequentially reacting and detecting the remaining five standard substances, wherein the detection conditions are as follows: excitation wavelength is 340 nm; receiving wavelengths 596nm and 615 nm; the delay time is 0.2 ms; the window time is 0.4 ms; cycle time 1.0 ms. The results are as follows: the working curve of the thyrotropin is shown in figure 2, the working curve has better linearity, and the detection method has good sensitivity and realizes the automation of time-resolved fluorescence detection by combining the numerical values in the table 1 and the figure 2.

TABLE 1 time-resolved fluoroimmunoassay for thyrotropin

Claims

1. A bipyridine derivative having a chemical formula:

2. the method for synthesizing the bipyridine derivative according to claim 1, wherein the bipyridine derivative is synthesized by a route comprising:

the method comprises the following steps:

A. epoxidizing a pyridine of the compound (4) with a peroxide to produce a compound (5);

D. reducing compound (7) to compound (8) with phosphorus tribromide;

E. condensing the compound (8) with 3-aminopropanol to produce a compound (9);

G. oxidizing the compound (10) with m-CPBA to compound (11);

H. reacting the compound (11) with acetic anhydride to produce a compound (12);

I. hydrolyzing the compound (12) to a compound (13);

J. brominating compound (13) to produce compound (1);

K. dissolving the compound (2) in a solvent containing lithium carbonate, reacting with the compound (1), stirring, cooling in an ice bath, filtering, evaporating the filtrate to dryness, and performing reverse phase chromatography on the residue to obtain a cryptate compound (3);

the structure of compound (2) is:

the structure of compound (3) is:

l, adding europium trichloride and the compound (3) into a solvent, refluxing, evaporating the solvent, dissolving in an acid solution, stirring at room temperature, then evaporating excess acid under reduced pressure, concentrating to dryness, and performing reverse phase chromatography to obtain the compound (3').

3. The method for synthesizing bipyridine derivatives according to claim 2, wherein the peroxide in step A is m-CPBA.

4. The method for synthesizing a bipyridine derivative according to claim 2, wherein the compound acylated with the compound (9) in the step F is benzoyl chloride.

5. The method of synthesizing a bipyridine derivative according to claim 2, wherein the bromination reagent in the J-step is phosphorus tribromide.

6. The method for synthesizing a bipyridine derivative according to claim 2, wherein the solvent is ethanol, methanol, ether, acetonitrile, dimethylsulfoxide, or dioxane.

7. The method for synthesizing a bipyridine derivative according to claim 2, wherein the acid solution is trifluoroacetic acid.

8. Use of the bipyridine derivative according to claim 1 for the preparation of a time-resolved fluorescent probe.

9. Use of the bipyridine derivative according to claim 1 for the preparation of a time-resolved fluorescence detection kit.